It is desirable to develop non-animal models for assessing cytotoxicity and genotoxicity of chemicals in humans. Accordingly, a human cell culture system offers the advantage of species identity and eliminates the need to extrapolate results from experiments in rodent or bacterial cells.
Metabolic activation is essential for the conversion of a wide variety of chemicals to ultimate cytotoxic or genotoxic agents. It is therefore desirable to provide a toxicological test system which is proficient in bioactivation of xenobiotics in order to reveal the toxicologic potential of an agent. Cytochromes P450 comprise the most important class of enzymes that bioactivate xenobiotics.
Cytochromes P450 are implicated in the activation of many cytotoxic agents, pro-mutagens and cancer chemotherapy agents. In particular, cytochrome P450IAI (CYP1A1) has been implicated in lung cancer by virtue of its ability to bioactivate components of cigarette smoke such as benzo(a)pyrene into potent mutagenic agents.
The prokaryotic test system most commonly used to detect mutagenicity at the present time is the Ames test (1). The Ames test is limited by the fact that bacteria used in the test lack bioactivating enzymes such as the cytochromes P450. Therefore the system cannot detect the mutagenicity of agents requiring metabolic activation.
A modified Ames test (2) has been developed which includes premetabolism by microsomal preparations. This test suffers from the fact that many agents, once metabolized to their active forms, are unable to cross the plasma membrane to enter the cells. In other words, the extracellular location of the enzymes greatly prejudices the effectiveness of the test. It is therefore further desirable to provide a test system consisting of mammalian cells metabolically competent for bioactivation.
There have been many approaches used by researchers to develop cell lines for use as test systems for bioactivation of chemicals. Each of these approaches are deficient in at least one aspect if not many aspects. With regard to these type of tests, there are three important considerations for developing engineered mammalian cell lines for examining bioactivation of chemicals and assessing their toxologic potential.
The first consideration is appropriate and stable expression of the chimeric gene construct. For example, Chinese hamster ovary cells have been transformed with rat cytochrome P450 IA1 (CYP1A1,(3)) and IIB1 (CYP2B1,(4)) cDNAs in an expression vector. The expression of the P450 CDNA in the vector is controlled by the strong, constitutive SV40 early promotor. One cell line had a few copies of the CYP1A1 but the other two had many copies. One of the high copy transformants produced several bands on genomic Southern blot strongly hybridizing with CYP1A1 probe suggesting rearrangement of the integrated genes. Multi-copy integration events are often unstable and the multi-copy CYP1A1 transgenes in these CHO lines are no exception (3). One of the lines is losing copies and the very high copy number transformant appears to be amplifying the transgene. Only one of the three is reported to be relatively stable. Only the relatively stable transformant and the low copy transformant show increased mutagenicity upon exposure to benzo(a)pyrene (BAP) and BAP-(trans)-7,8-diol, two compounds which are mutagenic only after metabolism by CYP1A1.
These studies show that expression of CYP1A1 or CYP2B1 resulted in an increased mutagenicity after exposure of the cells to compounds known to be bioactivated by these enzymes. However, the number of transformants obtained which remain stable in their expression of the P450 transgenes is few. It is hypothesized that this is due to the promotor used in the expression vector being a strong constitutive promotor in most situations. Constant, high levels of P450 expression can be deleterious to the cells. It is more desirable to have a P450 cDNA under control of an inducible promotor of moderate strength as opposed to the strong constitutive promotor used in the prior art.
The second consideration is the ability to draw conclusions about dose level in the human situation. One aspect of the problem is that human cells are more complete in their capability for DNA repair than are rodent cells which concentrate their repair efforts on transcriptionally active regions of the chromatin. Transformation of human cells with P450 cDNAs in expression vectors is more desirable for drawing conclusions about mutagenic doses in humans.
Other researchers have initiated studies of transfected cytochrome P450 cDNAs in human lymphoblasts (4,5,6). This system addresses the species specificity problem but does not allow for inducible expression of P450 as does the vector used to transform human cells in accordance with the present invention.
Very recent research has been directed to applying a mutant hamster cell line deficient in DNA repair to problems in environmental mutagenesis by introducing the mouse cytochrome P.sub.3 450 (P4501A2 subfamily) gene for metabolic activation of aromatic amines (6a). Others suggest an in vitro mutagenic system based on activation by human P450s to supplement other test systems (6b).
A third consideration is the ability to assess gene specific DNA damage. Comparison of DNA damage in specific genes is facilitated by using cells which do not repair any of the damage sites. For this reason, it is desirable to use human DNA excision repair deficient cells in addition to normal DNA repair proficient cells.